Iron is a required as a enzyme co-factor and as a substrate in the biosynthesis of heme. Excess cellular iron, however, leads to cell death and organ dysfunction. Iron can enter cells through the binding of iron laden transferrin to cellular transferrin receptors or through a second pathway which is independent of transferrin. The transferrin independent uptake of iron results from a membrane carrier that mediates the uptake of iron bound to low molecular weight chelators of "ionic iron". Iron uptake through this second pathway is responsible for the pattern of tissue iron overload. We propose to use biochemical and genetic approaches to isolate the transporter or its gene, and to determine whether this transporter is a transport system for other transition metals. Protocols are suggested that will allow for the selection of cultured cells with increased or decreased levels of transport activity. These mutants will be used to determine if there is a common transition metal transport system or if there are genetically separable systems. We also propose to isolate the cDNA for the ionic iron transport system using expression cloning in Xenopus oocytes. To examine the factors that regulate the intracellular distribu-tion of iron we propose to transfect cells with constructed genes which code for a shortlived protein whose synthesis is regulated by the concentration of free iron. Thus, the level of the protein is a measure of the size of the free iron pool. Using this probe we will examine factors that affect changes in the free iron pool. Such factors include an increase in the synthesis of the ferritin H chain as a result of exposure of cells to tumor necrosis factor, or exposure of cells to oxygen or nitrogen radicals. We also propose to study the physiology and mechanism of iron movement into and out of ferritin. We will test the hypothesis that iron release from ferritin requires the degradation of the intact molecule. To examine the movement of intracellular iron we will create an iron sink by introducing into cells vectors containing cDNA for ferritin H and L chains, in which the iron regulatory regions have been removed, and expression of the genes is regulated by an inducible promoter. We predict that ferritin synthesis may now reduce the free iron pool in cells resulting in cellular iron deprivation. These studies will yield information on the mechanisms of tissue iron overload and the role of iron in normal and pathological processes.